Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase
Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase
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DOI：
10.1128/jb.00986-15
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发表时间：
2016-06-01
影响因子：
3.2
通讯作者：
Shen, Aimee
中科院分区：
文献类型：
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作者：
Donnelly, M. Lauren;Fimlaid, Kelly A.;Shen, Aimee
艰难梭菌是世界各地抗生素相关性腹泻的主要原因。艰难梭菌要引起感染，其代谢休眠的孢子必须在胃肠道内萌发。在萌发过程中，孢子降解其保护皮层的肽聚糖层，释放二氢吡啶甲酸(DPA)，并使其核心水化。在艰难梭菌中，皮层水解物是DPA释放所必需的，而在枯草芽孢杆菌中，DPA释放是皮层水解物所必需的。鉴于这种差异，我们通过构建spVAC或dpaAB突变来测试艰难梭菌孢子萌发是否需要DPA合成和/或释放，这两个突变分别编码一个离子通道将DPA输送到前孔和预计合成DPA的酶复合体。艰难梭菌spVAC和dpaAB突变孢子缺乏DPA，但能稳定纯化，且比野生型孢子有更多的水份；相比之下，枯草杆菌spVAC和dpaAB突变孢子不稳定。虽然艰难梭菌的spVAC和dpaAB突变孢子表现出野生型萌发反应，但它们更容易在湿热下被杀死。皮层的水解不受这种处理的影响，这表明湿热抑制了这一事件下游的一个阶段。有趣的是，艰难梭菌spVAC突变孢子对热处理的敏感性明显高于dpaAB突变孢子，这表明SpoVAC在赋予耐热性方面起着额外的作用。综上所述，我们的结果表明，SpoVAC和DPA合成酶控制着艰难梭菌的孢子耐药性，并揭示了不同细菌对这些蛋白质的不同要求。艰难梭菌是一种形成孢子的专性厌氧菌，在美国每年导致大约50万例感染。虽然孢子萌发是艰难梭菌致病的关键因素，但这一过程所需的因素只有部分特征。本研究描述了两个因子DpaAB和SpoVAC的作用，这两个因子分别控制细菌孢子合成和释放二吡啶甲酸(DPA)。以前在其他孢子形成生物中对这些蛋白质的研究表明，它们对孢子形成、萌发和抗性是不同的需要。我们现在证明，这些蛋白质对于艰难梭菌的孢子形成和萌发是必不可少的，但对于耐热性是必要的。因此，我们的研究进一步强调了DpaAB和SpoVAC在孢子形成生物中的不同功能。
The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes.IMPORTANCEClostridium difficile is a spore-forming obligate anaerobe that causes similar to 500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid ( DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.